Graminaceous plants that absorb by chelating the insoluble state Fe(III) in soil using mugineic acid and adopt so called the Strategy-II mechanism of Fe acquisition secrete Fe chelators (phytosiderophores) from their roots to solubilize sparingly soluble Fe in the rhizosphere (Roemheld, 1987). The amount of the secreted phytosiderophores increases under Fe-deficiency stress. The mugineic acid family is the only examples of phytosiderophores known so far (Takagi, 1976). Tolerance to Fe deficiency in graminaceous plants is thought to depend on a quantity of mugineic acid family secreted by plants (Takagi et al. 1984, Roemheld and Marschner 1986, Marschner et al. 1987, Mori et al. 1987, Kawai et al. 1988, Mori et al. 1988, Mihashi and Mori 1989, and Shingh et al. 1993).
The biosynthetic pathway of mugineic acid in plants is shown in FIG. 1. S-adenosylmethionine is synthesized from methionine by S-adenosylmethionine synthase. Subsequently, three molecules of S-adenosylmethionine are combined to form one molecule of nicotianamine by nicotianamine synthase. The generated nicotianamine is then converted to 3″-keto acid by nicotianamine aminotransferase, and 2′-deoxymugineic acid is synthesized by the subsequent action of a reductase. A further series of hydroxylation steps produces the other mugineic acid derivatives including mugineic acid from the deoxymugineic acid (Mori and Nishizawa 1987, Shojima et al. 1989, Shojima et al. 1990 and Ma and Nomoto 1993).
A compound in FIG. 1, a compound in the lower right, wherein R1 and R2 are hydrogen and R3 is hydroxyl, is mugineic acid. A compound wherein R1 is hydrogen and R2 and R3 are hydroxyl, is 3-hydroxymugineic acid. Also a compound wherein R2 is hydrogen and R1 and R2 are hydroxyl, is 3-epihydroxymugineic acid.
Three S-adenosylmethionine synthase genes were isolated from barley roots, but these genes were not induced by Fe deficiency (Takizawa et al. 1996). A gene Ids3, which is obtained from the barley by differential screening, is suspected to be a gene, which converts deoxymugineic acid to mugineic acid by hydroxylation and is strongly induced by Fe-deficiency (Nakanishi et al. 1993). Further, nicotianamine aminotransferase was purified and isolated from Fe-deficient barley roots, and two nicotianamine aminotransferase genes, Naat-A and Naat-B, were isolated (Takahashi et al. 1997). Naat-A expression was induced in Fe-deficient roots.
The synthesis of nicotianamine from S-adenosylmethionine is similar to polyamine synthesis from decaroboxy-S-adenosylmethionine. In contrast to polyamine synthase, however, nicotianamine synthase catalyzes the combination of three S-adenosylmethionine molecules and the azetidine ring formation at the same time (FIG. 1). Such the nicotianamine synthase is a novel type of enzyme. Previously, we reported the partial purification of nicotianamine synthase from the roots of Fe-deficient barley and expression pattern of the activity (Higuchi et al. 1994, Higuchi et al. 1995, Kanazawa et al. 1995, Higuchi et al. 1996a and Higuchi et al. 1996b). Since nicotianamine synthase is easily decomposed during extraction and purification, it has been difficult to purify sufficient quantities for amino acid sequencing.
The present invention has an object to provide a plant, especially graminaceous plant, highly tolerant to Fe-deficiency, as a result of isolating and purifying a nicotianamine synthase, being cloned the gene of this enzyme, determining the base sequence and amino acid sequence thereof, and using said enzyme.